A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin.

Abstract

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.

Neighbor-joining tree of mouse SET domain proteins. Sequences of mouse SET domain proteins were identified from public databases. SET domains sequences were aligned, and a neighbor-joining tree showing related SET domain proteins was constructed. Homologous protein sequences in Drosophila and S. pombe were identified by blast searches. HMTase specificity of subtrees was assigned according to the described HMTase activity of representative enzymes, such as H3-K4 (Ash1 [], Mll [], Set7/9 [; ]); H3-K9 (Suv39h1 [], ESET [], G9a []); H3-K27 (Ezh2 [; ]); H3-K36 (Nsd1 []); and H4-K20 (Pr-Set7 [; ]). Proteins that were selected for the candidate approach are indicated by an asterisk.

Identification of Suv4-20h1 and Suv4-20h2 as heterochromatic H4-K20 trimethylating HMTases. (A) Protein structure of Suv4-20h proteins. The SET domain is located in the N terminus and flanked by N-as well as C-terminal extensions of high sequence homology within Suv4-20h orthologous proteins. In addition, there is a conserved region in the C terminus of these proteins. (B) EGFP-tagged Suv39h1, Suv4-20h1, and Suv4-20h2 were expressed in wild-type and Suv39h double-null (dn) female MEFs. After fixation, distribution of EGFP-tagged proteins was analyzed by fluorescence microscopy. DAPI dense foci represent pericentric heterochromatin. (C) RNAi mediated knock-down of Suv39h and Suv4-20h proteins. Female wild-type MEFs were cotransfected with pEGFP-N1 as transfection marker and pSUPER vector expressing hairpin oligos directed against Suv39h1/Suv39h2 or Suv4-20h1/Suv4-20h2. After 5 d in selection medium, cells were stained with α-trimethyl H3-K9 and α-trimethyl H4-K20 antibodies. Immunostaining was examined in EGFP-positive cells.

Definition of a heterochromatic targeting module in Suv4-20h2. (A) Various truncations of Suv4-20h2 (S#1-S#6) were expressed as EGFP fusion proteins in female wild-type MEFs. Cells were fixed, and distribution of fusion proteins was examined with fluorescence microscopy. DAPI dense foci represent pericentric regions. Note, that EGFP foci of subfragment S#4 do not represent pericentric heterochromatin, because they do not colocalize with DAPI dense regions. (B) GST-tagged protein truncations of Suv4-20h2 (S#1-S#6) as well as recombinant GST were bound to glutathione-Sepharose and incubated with in vitro translated myc3-tagged HP1α, HP1β, and HP1γ. After extensive washing steps, bound proteins were separated on SDS-PAGE, and Western blots were probed with α-myc antibodies. (C) Summary table showing structure of Suv4-20h2 protein truncations together with localization and HP1 interaction data. (HET) Heterochromatin; (EU) euchromatin aberrant.